Glycosylation in eukaryotes has been studied intensively for decades as the most common covalent post translational protein modification mechanism. About 1-2% of the human transcriptome (about 250-500 glycogenes) is predicted to translate proteins which are responsible for glycosylation (Campbell and Yarema 2005). Glycosylation of cellular proteins plays many key biological functions such as protein folding, stability, intracellular and inter-cellular trafficking, cell-cell and cell matrix Interaction.
There are four distinct groups of Glycoproteins: N-linked, O-linked, glycosaminoglycans, and glycosylphosphatidylinositol-anchored proteins. N-linked glycosylation occurs through the side chain amide nitrogen of asparagine residues, while O-linked glycosylation uses the oxygen atom in the side chain of serine or threonine residues. N-linked glycosylation takes place in the amino acid sequence of Asn-X-Ser/Thr, where X can be any amino acid except proline and aspartic acid (Helenius and Aebi 2004).
Fucose (6-deoxy-L-galactose) is a monosaccharide that is present in many glycoproteins and glycolipids present in vertebrates, invertebrates, plants, and bacteria. Fucosylation is the process of transferring a fucose residue to various proteins and oligosaccharides. Fucosylation is regulated by several molecules, including fucosyltransferases, guanosine diphosphate (GDP)-fucose synthetic enzymes, and GDP-fucose transporter(s). A large number of fucosylated glycoproteins are secretary proteins or membrane proteins on the cell surface.
There are 14.1 million new cancer cases, 8.2 million cancer deaths and 32.6 million people living with cancer (within 5 years of diagnosis) in 2014 worldwide. The high mortality rate of cancer serves as a reminder of the need for more effective therapies. The most prominent change in oncology drug development in the last 20 years has been the shift from classic cytotoxics to drugs that affect signaling pathways implicated in cancer, known as “Monoclonal Antibodies” or mAbs. A decade ago, there are only two mAbs on the market and currently there are around 30 FDA approved mAbs of diverse therapeutic modalities, like Adalimumab, Infliximab, Rituximab etc. mAbs are the fastest growing segment in pharmaceutical industry and this rapid expansion is set to continue. There are more than 100 monoclonal antibody-based biologic drugs in clinical trials. Many of these are in phase II and phase III trials and will be coming before the Regulatory agencies for approval. Improvement of monoclonal antibody therapeutics through technologies described here will pave the way for better clinical outcome for patients.
Human IgG1 antibody is a highly fucosylated glycoprotein. Two N-linked biantennary oligosaccharides consisting of core hepta-saccharide with variable addition of fucose, galactose, bisecting N-acetylglucosamine and sialic acid are present at Asn-297 of IgG. Antibody glycosylation leads to unique biological functions known as “effector functions”—Antibody Dependent Cellular Cytotoxicity (ADCC) and Complement Dependent Cytotoxicity (CDC). ADCC is a cell mediated immune system where immune cells (like natural killer cells) lyse the target cells identified through antibodies against cell surface antigens.
The effector function of IgG molecule is defined by the interaction of antibody Fc region with leukocyte receptors, known as FcγRs, or interactions with complement components. The composition of the oligosaccharide structure is critically important for effector function through FcγR binding (Shields et al. 2002; Shinkawa et al. 2003; Niwa et al. 2004; Niwa, Shoji-Hosaka, et al. 2004; Yamane-Ohnuki et al. 2004). Crystal structure analysis of human IgG1 has revealed intricate interaction of the oligosaccharide chains with the CH2 domain (Harris et al. 1998; Radaev et al. 2001).
The efficiency of the ADCC mechanism is considerably dependent on the level of antibody fucosylation. The lower the fucosylation, the higher is the rate of ADCC. Therefore, loss of fucosylation has significant biological consequences. The loss could be due to non-functional fucosyltransferase enzymes, resulting in non-fucosylation of cellular proteins. The absence of fucose from the primary N-acetylglucosamine results in the IgG1 antibody having increased binding affinity for the FcγRIIIα receptor, with consequent increase of 50-100 times higher efficacy of ADCC. Improvement of ADCC with non-fucosylated IgG is directly proportional to the increased affinity for FcγRIIIα which allows the non-fucosylated IgG Fc to overcome the competition from high concentrations of fucosylated IgG in normal serum. Plausible rationale for the increased affinity of non-fucosylated IgG Fc for FcγRIIIα may be the reduction or absence of steric inhibition at the receptor-ligand interface (Harris, 1998; Radaev, 2001).
In mammalian expression system, the enzyme α1,6-fucosyltransferase encoded by the Fut8 gene is responsible for transferring fucose moiety from GDP-fucose to N-acetylglucosamine of N-glycan chain in proteins (Miyoshi, 1999). Disruption of this gene function through various means leads to production of non-fucosylated proteins, including antibodies (Naoko Yamane-Ohnuki, 2004).
GDP-D-mannose 4,6-dehydratase (GMD) is a member of the sugar nucleotide-modifying subfamily of the short-chain dehydrogenase/reductase (SDR) family (Webb, Mulichak et al. 2004).
In mammalian expression systems, GDP-fucose, an essential substrate of fucosylation, is synthesized in the cytoplasm through de novo and salvage pathways. In the de novo pathway of fucosylation, GDP-fucose is synthesized through conversion of GDP-mannose to GDP-4-keto-6-deoxy-mannose, catalyzed by the enzyme GDP-mannose 4,6-dehydratase (GMD). This GDP-Fucose is then transported inside the golgi and used as a substrate for protein fucosylation by the enzyme al-6 fucosyltransferase (FUT8). The enzyme transfers the fucose moiety from GDP-fucose to N-acetylglucosamine of the N-glycan chain (Miyoshi, 1999). These critical enzymes, GDP-mannose 4,6-dehydratase and α,1-6 fucosyltransferase are encoded by GMD and FUT8 genes respectively.
Non-fucosylated forms of therapeutic antibodies developed in mammalian platforms, where fucose biosynthesis is impaired, may have clinical advantage over the fucosylated forms due to the enhanced efficiency of ADCC towards target tumor cells.
Historically, gene knock out systems completely depended on Homologous recombination (HR) mediated targeted mutation, deletion and/or insertion. The HR system, although very specific, is highly inefficient, as thousands of clones need to be screened to find one mutated clone. Moreover, deleting allelic variations would take even further time and much larger screening. Multiple technologies have evolved in the last decade to achieve targeted gene modification using a combination of a DNA sequence recognition domain and a nuclease domain. These systems are highly efficient at identifying specific sites of interest and then introducing DNA strand breaks. DNA double-strand break (DSB) at genomic target locus activates DNA repair, which is utilized for modifying genes. The DNA damage response is highly conserved in eukaryotic cells. The concept of DSB-based genome engineering is easily transferable between highly diverse organisms. Creating double strand break increases the frequency of gene knock out at targeted loci by thousand folds through homologous recombination and non-homologous end joining mechanisms.
Zinc Finger Nuclease (ZFN) is one of the most frequently used techniques for gene disruption. It requires three bases at the DNA level for each zinc finger tandem array. Moreover, target site overlap and cross-talk between individual fingers in a zinc-finger array considerably complicate the production of sequence-specific ZFNs. Additionally, major drawback of ZFNs includes elaborate and time-consuming experimental selection process to identify the ZFN motifs for specific DNA sequence recognition.
There are methods in the prior art for disruption of Fut8 and GMD genomic loci. However, none of the methods target the specific location on the FUT8 and GMD genomic loci by the CRISPR technology.
The present disclosure overcomes the disadvantages or limitations associated with methods of the prior art by using the CRISPR technology to target a specific location on the FUT8 genomic loci or the GMD genomic loci, which results in complete disruption of the gene and related function, providing a cell that produces non-fucosylated proteins.